title: Construction of a heterologous gene expression system in the banana rhizobacterium strain GW-3 and its colonization ability.
abstract: Rhizobacteria inhabiting the rhizosphere are beneficial to their host plants, and can potentially serve as biocontrol agents to control plant diseases. We isolated the rhizobacterium strain GW-3, which was the dominant bacterium in the rhizosphere soils of healthy banana plants. Then, we constructed an expression system with a kanamycin resistance gene to express a heterologous protein in GW-3. Using the green fluorescent protein gene as the reporter, we monitored expression of the heterologous protein by detecting fluorescence intensity and conducting western blot analyses. The standard fluorescence intensity of the recombinant strain reached 1,482 ± 3.49 RFU. To study the colonization ability of GW-3, we inoculated this bacterium into sterilized and unsterilized rhizosphere soils and monitored the bacterial population over 25 days. The populations of GW-3 in rhizosphere soils first increased, then decreased, and finally reached a balance. Laser scanning confocal microscope analyses of fluorescence in banana roots after inoculation with GW-3 confirmed that the recombinant GW-3 strain stably colonized banana root surfaces. Analyses of the bacterial population in unsterilized rhizosphere soils showed that the recombinant GW-3 strain was still the dominant bacterium in banana rhizosphere soils at 25 days after inoculation. Together, these results showed that this expression system can be used to express a heterologous protein at high levels in a dominant rhizobacterium. By incorporating relevant resistance genes into the expression system, this method could be used to genetically engineer GW-3 to control banana wilt disease.
authors.affiliation: Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, China.
authors.forename: Yuguang
authors.initials: Y
authors.lastname: Wang
authors.forename: Qiyu
authors.initials: Q
authors.lastname: Xia
authors.forename: He
authors.initials: H
authors.lastname: Zhang
authors.forename: Xuehua
authors.initials: X
authors.lastname: Lu
authors.forename: Jianbo
authors.initials: J
authors.lastname: Sun
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
mesh_terms: D015153:Blotting, Western; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal; D008967:Molecular Biology / Q000379:methods*; D028521:Musa / Q000382:microbiology*; D018517:Plant Roots / Q000382:microbiology; D012230:Rhizobiaceae / Q000235:genetics* / Q000254:growth & development*
chemical_list: D049452:Green Fluorescent Proteins